*/Disease Information(sort of like the Intro to your MiniResearch Writeup): Methanocaldococcus jannaschii is part of the archaea domain and are single celled organisms. They are found in extreme environments such as the hypothermal vents at the bottom of the ocean where water boils and has high pressure. This organism has been found to have the same efflux pumps and permeases that transport hydrophobic drugs out of certain bacteria causing the bacteria not to be affected. If they have these same pumps/permeases, then M. jannaschii would be able to be unaffected by those drugs and cause harm to humans if the organism invaded.
--- List cost and quantity of substrate reagents, supplier, and catalog #
GMPS can be assayed via dye binding with bovine serum albumin standard and a spectrophotometer. Bradford reagent contains Brilliant Blue G in phosphoric acid and methanol. To assay, you mix one part protein to thirty part reagent and run it through the spectrophotometer and recorded at absorbance of 595 nm. The Sigma Aldrich catalog number for the reagent is B6916 and it costs $70.00 for 500 ml.
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2LXN
Current Inhibitors:none directly for Methanocaldococcus jannaschii. For expression in E. coli, inhibitors include 2-fluoroinosine 5’-monophosphate, 1-methyladenosine, and 2’-deoxyadenosine.
Expression Information (has it been expressed in bacterial cells):expressed in E. coli
Purification Method:GMP synthase can be purified by having a 4M solution of the protein denaturant guanidinium thiocyanate and .1 M 2-mercaptoethanol to break the disulfide bonds. The GMP can be filtered by ethanol precipitation or sedimentation with cesium chloride.
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'): MIVILDNGGQYVHRIHRSLKYIGVSSKIVPNTTPLEEIESNKEVKGIILSGGPDIEKAKN CIDIALNAKLPILGICLGHQLIALAYGGEVGRAEAEEYALTKVYVDKENDLFKNVPREFN AWASHKDEVKKVPEGFEILAHSDICQVEAMKHKTKPIYGVQFHPEVAHTEYGNEILKNFC KVCGYKFE
*length of your protein in Amino Acids:188
Molecular Weightof your protein in kiloDaltons using the**Expasy ProtParam**website: 21020
MolarExtinction coefficientof your protein at 280 nm wavelength:17420
*NCBI Gene # or RefSeq#: WP_010871100.1
*Organism (including strain): Methanocaldococcus jannaschii
Etiologic Risk Group (see link below): n/a
*/Disease Information(sort of like the Intro to your MiniResearch Writeup):
Methanocaldococcus jannaschii is part of the archaea domain and are single celled organisms. They are found in extreme environments such as the hypothermal vents at the bottom of the ocean where water boils and has high pressure. This organism has been found to have the same efflux pumps and permeases that transport hydrophobic drugs out of certain bacteria causing the bacteria not to be affected. If they have these same pumps/permeases, then M. jannaschii would be able to be unaffected by those drugs and cause harm to humans if the organism invaded.
Link to TDR Targets page (if present):
Link to Gene Database page (NCBI, EuPath databases -e.g. TryTryp, PlasmoDB, etc - or PATRIC, etc.): https://www.ncbi.nlm.nih.gov/protein/WP_010871100.1
Essentiality of this protein: GMP synthase is used to synthesize GMP from XMP which is a part of purine metabolism.
Is it a monomer or multimer as biological unit? (make prediction at http://www.ebi.ac.uk/msd-srv/prot_int/pistart.html): Monomer
Complex of proteins?: no
Druggable Target (list number or cite evidence from a paper/database showing druggable in another organism): not available
*EC#: 6. 3. 5. 2
Link to BRENDA EC# page:http://www.brenda-enzymes.org/enzyme.php?ecno=6.3.5.2
-- Show screenshot of BRENDA enzyme mechanism schematic
Enzyme Assayinformation (spectrophotometric, coupled assay ?, reagents):
-- link to Sigma (or othercompany) page for assay (see Sigma links below)
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf
-- -or link (or citation) to paper that contains assay information https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3859818/#R38
-- links to assay reagents (substrates) pages.
https://www.sigmaaldrich.com/catalog/product/sigma/b6916?lang=en®ion=US
--- List cost and quantity of substrate reagents, supplier, and catalog #
GMPS can be assayed via dye binding with bovine serum albumin standard and a spectrophotometer. Bradford reagent contains Brilliant Blue G in phosphoric acid and methanol. To assay, you mix one part protein to thirty part reagent and run it through the spectrophotometer and recorded at absorbance of 595 nm. The Sigma Aldrich catalog number for the reagent is B6916 and it costs $70.00 for 500 ml.
Structure (PDB or Homology model)
-- PDB # or closest PDB entry if using homology model: 2LXN
Current Inhibitors: none directly for Methanocaldococcus jannaschii. For expression in E. coli, inhibitors include 2-fluoroinosine 5’-monophosphate, 1-methyladenosine, and 2’-deoxyadenosine.
Expression Information (has it been expressed in bacterial cells): expressed in E. coli
Purification Method: GMP synthase can be purified by having a 4M solution of the protein denaturant guanidinium thiocyanate and .1 M 2-mercaptoethanol to break the disulfide bonds. The GMP can be filtered by ethanol precipitation or sedimentation with cesium chloride.
Image of protein
[[image:blob:https://vdsstream.wikispaces.com/d5522b55-52cd-4334-ac5e-5893927e199a]]
Figure 1. Structure of 2LXN from Methanocaldococcus jannaschii
*Amino Acid Sequence (paste as text only - not as screenshot or as 'code'):
MIVILDNGGQYVHRIHRSLKYIGVSSKIVPNTTPLEEIESNKEVKGIILSGGPDIEKAKN
CIDIALNAKLPILGICLGHQLIALAYGGEVGRAEAEEYALTKVYVDKENDLFKNVPREFN
AWASHKDEVKKVPEGFEILAHSDICQVEAMKHKTKPIYGVQFHPEVAHTEYGNEILKNFC
KVCGYKFE
*length of your protein in Amino Acids: 188
Molecular Weightof your protein in kiloDaltons using the**Expasy ProtParam**website: 21020
MolarExtinction coefficientof your protein at 280 nm wavelength: 17420
TMpred graph Image (http://www.ch.embnet.org/software/TMPRED_form.html). Input your amino acid sequence to it.
Figure 2. TMpred output for 2LXN in Methanocaldococcus jannaschii
*CDSGene Sequence(paste as text only): ATGATTGTTATCTTAGACAACGGAGGGCAGTATGTTCATAGAATACACAGAAGTTTAAAG
TATATTGGAGTTAGCTCAAAGATAGTTCCAAATACAACACCATTGGAAGAGATTGAAAGC
AATAAAGAAGTTAAGGGTATTATACTAAGTGGAGGGCCTGATATTGAAAAAGCTAAAAAT
TGTATAGATATTGCTTTAAATGCTAAACTCCCTATTTTGGGGATTTGTTTAGGACATCAA
TTAATTGCCTTGGCTTATGGTGGAGAGGTTGGAAGGGCTGAGGCAGAAGAATACGCACTA
ACAAAGGTCTATGTAGATAAAGAAAACGATTTATTTAAAAACGTTCCAAGAGAGTTCAAT
GCATGGGCTTCACATAAGGATGAGGTTAAAAAAGTTCCAGAAGGTTTTGAGATTTTAGCT
CATTCAGATATATGTCAGGTTGAAGCAATGAAACATAAAACAAAGCCGATTTATGGAGTT
CAGTTCCACCCTGAAGTTGCTCATACAGAATATGGAAATGAGATTTTGAAAAACTTTTGT
AAAGTTTGTGGTTACAAATTTGAATAA
*GC% Content for gene: 34.39%
*CDS Gene Sequence (codon optimized) - copy fromoutputofPrimer DesignProtocol (paste as text only): n/a
*GC% Content for gene (codon optimized): n/a